Early prenatal diagnosis to detect fetal genetic disorders is desirable for both expectant mothers and physicians to make informed decisions. Definitive methods of invasive prenatal testing (amniocentesis and chorionic villous sampling) carry a small, but significant risk of miscarriage, and the results are rarely available before 13 weeks of pregnancy because of the time required for cell culture and analysis.
“Non-invasive” screening with maternal serum analyte screening and ultrasound can identify individuals at risk for fetal aneuploidy (predominantly trisomy 21), but a positive screening result still requires a subsequent invasive procedure for a definitive diagnosis. Of some 25-30 such procedures, only one will actually show a fetal aneuploidy.
Many laboratories around the world have been attempting for over a decade to develop non-invasive (i.e. venupuncture only) methods to isolate and analyse fetal cells. An obvious advantage is that definitive results can be obtained using molecular techniques such as fluorescence in-situ hybridization (FISH) and quantitative fluorescent polymerase chain reaction (QF-PCR) on recovered fetal cells.
The presence of fetal cells in maternal blood provides potentially the best possible source of cells for non-invasive prenatal diagnosis. However fetal cells are present at very low numbers, and their isolation is not a trivial task, with only 1 or 2 fetal cells being present per 10 ml maternal blood. Evidence also indicates that the presence of intact fetal cells in the maternal circulation is not a universal event.
An alternative to peripheral blood sampling is the isolation and analysis of trophoblasts from transcervical samples. Unlike maternal blood in which multiple circulating fetal cell types exist, fetal cells in the transcervical samples are all of placental origin and are overwhelmingly trophoblasts (Bischoff and Simpson, 2006).
It was long assumed that the cervical canal contained trophoblasts of fetal origin. The early embryo is covered with chorion levae, but later in the gestation the chorionic surface is smooth. However, it was not until 1971 that the presence of fetal cells in the endocervix was confirmed by identification of Y-chromosome bearing cells in midcervical mucous samples collected with a cotton swab (Shettles et al., 1971). Subsequent reports assumed that these fetal cells were shed from the regressing chorionic villous into the lower uterine pole (Warren et al., 1972, Adinolphi et al., 1995, Rhine et al., 1975). In this scenario, it is most likely to occur between 7 and 13 weeks gestation, before fusion of the deciduas basalis and parietalis. Desquamated trophoblasts are believed first to accumulate behind the cervical mucous at the level of the internal opening section (Bulmer et al., 1995, Adinolphi and Sherlock, 1997) and then become ensconced in the cervical mucous.
These biologic events thus define the window of opportunity for endocervical sampling to be of use for prenatal diagnoses, although several studies have demonstrated trophoblast recovery as early as 5 weeks gestation (Katz-Jaffe et al., 2005, Mantzaris et al., 2005).
Efforts to extract trophoblasts were first made in the 1970's. Rhine et al. (1975 and 1977) described “antenatal cell extractors” that flush the endocervical canal with sterile saline to recover fetal cells. After culture, fetal metaphases from recovered cells were detected in approximately 50% of cases. However, other investigators reported negative results (Goldberg et al., 1980), leading to overall skepticism concerning clinical application.
Interest was rekindled in the 1990's following the introduction of chorion villus sampling (CVS). Transcervical specimens were collected by cotton swabs, cytobrush, aspiration of cervical mucus with a catheter, lavage of the endocervical canal or uterine. A variety of techniques resulted in detection of fetal cells in 40-90% of specimens examined (Adinolfi et al., 1995a, Bussani et al., 2002, Cioni et al., 2003, Fejgin et al., 2001, Massari et al., 1996; Miller et al., 1999; Rodeck et al., 1995; Tuttschek et al., 1995). Again, however, interest waned in most centres because analysis was difficult. The presumptive fetal cells embedded in mucous were not readily amenable to FISH. More recently, molecular PCR techniques for micromanipulated cell clumps of trophoblastic origin were demonstrated to have utility for transcervical samples (Bussani et al., 2004; Bussani et al., 2007; Katz-Jaffe et al., 2005).
Most transcervical specimens contain a variety of maternally derived cells (leukocytes, macrophages, squamous epithelia, columnar epithelia, and endocervical cells) as well as different fetal-derived cells (cytotrophoblasts and syncytiotrophoblasts) (Bulmer et al., 1995, Miller et al., 1999). The frequency of each fetal cell type is variable and seemingly dependent on the collection method and gestational age.
The literature is inconsistent with regard to the number and relative proportion of fetal cells which can be recovered in transcervical specimens. Kingdom et al. (1995) reported the frequency of fetal XY cells recovered endocervical lavage to range from 2 to 8%. In the same study, FISH results using a cytological brush ranged from 1 to 5% of total cells. Daryani et al. (1997) reported fetal cells to be 3.6 to 47.8% of total cells, based on 3-31 fetal cells obtained by aspiration. Katz-Jaffe et al. (2005) claimed a higher absolute number of fetal cells, up to 250 cells/ml of dissociated mucous, based on immunohistochemistry staining with trophoblast specific monoclonal antibodies (NDOG1 and FT141.1).
Nuclei have previously been isolated from cells and tissues, usually by detergent treatment, or enforced lysis of cells by ammonium chloride to liberate nuclei from intact cells (for example see, Hymer and Cuff 1963; Antalis and Godbolt 1991; Krishan and Dandekar 2005). U.S. Pat. No. 5,447,864 discloses a method for isolating cell nuclei via selective lysis of the cell plasma membranes leaving the majority of nuclear membranes intact. U.S. Pat. No. 4,906,561 discloses a method for isolating cell nuclei by detergent lysis with simultaneous fluorescent labelling of nuclei for subsequent cytometric analysis. EP 0944829 and WO 98/026284 disclose a method for selectively lysing fetal nucleated red blood cells within a blood sample obtained from pregnant women by saponin mediated cell lysis.
There is a need for alternate methods of enriching fetal genetic material from a pregnant female.